Nutritional Composition for Improving Muscle Function and Daily Activity

ABSTRACT

The invention relates to a nutritional composition comprising
     (a) at least 18 en % of proteinaceous matter, said proteinaceous matter comprising whey;   (b) at least 12 wt % of leucine, based on total proteinaceous matter; and   (c) a lipid fraction comprising at least a ω-3 polyunsaturated fatty acid selected from the group of eicosapentaenoic acid, docosahexaenoic acid, eicosatetraenoic acid and docosapentaenoic acid for improving the muscle function in a mammal, for improving daily activity, for improving physical performance, for providing a better prognosis in terms of extended life-expectancy, for improving compliance to an anti-cancer therapy or for improving a quality of life.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 12/980,727 filed Dec. 29,2010 which is a continuation of PCT application number PCT/NL2009/050395designating the United States and filed Jul. 2, 2009; which is acontinuation in part of PCT application number PCT/NL2008/050447 andfiled Jul. 2, 2008, both of which are hereby incorporated by referencein their entireties.

DESCRIPTION

The invention relates to a composition comprising proteinaceous matterthat comprises leucine and an ω-3 polyunsaturated fatty acid.

The invention also relates to the use of a composition suitable forimproving the muscle function of a mammal. The invention also relates tothe use of a composition suitable for improving daily activity of amammal.

BACKGROUND

Body composition, muscle functionality and daily activity are clinicallyhighly relevant parameters, because muscle function and daily activityare important contributors to the quality of life of a cancer patient.Furthermore, an improved physical condition of a patient, as reflectedin body composition and physical performance, might contribute to itscompliance to an anti-cancer therapy. E.g. the dose of chemotherapy canbe provided as scheduled, instead of being adjusted to a lower bodyweight of the patient.

Cachexia is one of the most debilitating aspects of cancer and has beenassociated with increased morbidity and mortality, with a reducedquality of life, an impaired response to chemotherapy, an increasedsusceptibility to chemotherapy-induced toxicity and higher incidence ofpostoperative complications. Cancer cachexia can be defined asinvoluntary weight loss with a depletion of not only fat mass but alsolean body mass due to muscle wasting. Symptoms besides mass loss aredebilitation, weakness, edema, an impaired immune response and declineof motor and mental function. Cachectic patients have been shown to havehigher resting energy expenditure which is not met by an increasednutrient intake, in many cases food intake is even reduced. Hence,approximately 45% of cancer patients loose more than 10% of theirpre-diagnostic mass. The tumor can induce metabolic changes in proteinmetabolism, resembling those found in infection or injury. These changesare characterized by net protein breakdown and increased oxidation ofbranched-chain amino acids (BCAAs) in muscle to support energy supplyand synthesis of gluconeogenic amino acids. The breakdown of hostprotein is partly stimulated by inflammatory mediators produced by thehost (e.g. TNFα, IL-6)(1), but also by the tumor, through the release ofa proteolysis inducing factor (PIF)(4). Moreover, the tumor has a highintrinsic protein synthesis rate and has the capacity for intracellulartransport and catabolism of BCAAs.

An increased energy demand and an inflammatory catabolic status leads toan important reduction in body fat content and more worse muscle mass.It is therefore, hypothesized that nutritional support in cancerpatients should aim more for counteracting net body protein breakdown,than for merely increasing caloric intake per se. In order to establisha new, positive balance in protein synthesis and breakdown,supplementation of protein should be combined with components modifyingand mitigating the catabolic signal. A high amino acid supply has beendescribed to be essential for increasing protein synthesis. BCAAs andespecially leucine are known to control skeletal muscle proteinmetabolism by stimulating protein synthesis and inhibiting proteinbreakdown. Prospective caloric- and nitrogenous-controlled trials ofBCAA supplementation via TPN in septic patients indeed resulted in animprovement of pre-albumin levels and decreased overall mortality in apatient group with a high Simplified Acute Physiology Score(LeGall-SAPS) classification. Supplementation of tumor-bearing rats witha diet supplemented with 3% leucine has been reported to reduce loss oflean body mass, gastrocnemius muscle mass and myosin content, whencompared to an isonitrogenous and isocaloric control diet. These dataare supported by the observation that leucine increased proteinsynthesis in pregnant tumor-bearing rats, possibly resulting fromchanges in the ubiquitin-proteasome system. Two clinical trials studiedoral BCAA supplementation after surgical removal of the tumor andreported a shorter hospital stay, a better performance status at 3months and an increased body mass at 1 year. BCAAs have also beensupplemented in the presence of the tumor: patients undergoingchemotherapy received oral BCAA supplementation up to 1 year, resultingin a lower overall morbidity, improved nutritional status and betterquality of life. Other nutrients which may have anti-cachectic effectsare ω-3 polyunsaturated fatty acids (PUFAs). The vast majority of theclinical trials in which ω-3 PUFAs were tested report an increase ormaintenance of body mass (BW); while in two clinical trials no effect onthe loss of BW was found. In the latter, however, the supplementationperiod was only 2 weeks and/or included only a small number of patients.Other effects of EPA or fish oil supplementation in cancer patients,were a net lean tissue gain, an increase in total resting energyexpenditure and physical activity level, a decrease in need for TPN andan improved quality of life, and even suggested improved survival.

WO 2004/026294 discloses nutritional compositions comprising a mixtureof essential amino acids in free form and/or in salt form, rather thanintact protein, for the promotion of muscle protein synthesis orcontrolling tumor-induced weight loss, such as cancer cachexia. Intactprotein may be present in addition. However, a nutritional compositioncomprising at least 18 en % proteinaceous matter, at least part of whichis whey protein, at least 12 wt. % leucine and an ω-3 polyunsaturatedfatty acid selected from the group of eicosapentaenoic acid,docosahexaenoic acid, eicosatetraenoic acid and docosapentaenoic acid isnot described in a single combination. Exemplified nutritionalcompositions comprise caseinate as protein source. In Example 2 of WO2004/026294, it is concluded that ingestion of free essential aminoacids is more effective than ingestion of a comparable amount of intactprotein in stimulating net muscle protein synthesis.

EP 1 774 973 A1 discloses a composition comprising proteinaceous matter,said proteinaceous matter providing at least 24 en % and at least 12 wt% of leucine, based on total proteinaceous matter, for the treatment ofinsuline resistance. No compositions were exemplified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the differences in muscle Tibialis Anterior mass(FIG. 1A) and Epididymal fat mass after different interventions (FIG.1B).

FIGS. 2A, 2B, 2C, and 2D graphically depict the skeletal musclefunction: force frequency curve (ex-vivo).

FIGS. 3A, 3B, 3C, and 3D graphically depict the skeletal muscle functionduring exercise (ex-vivo). FIGS. 4A, 4B, 4C, and 4D depict total dailyactivity.

FIG. 5 depicts muscle protein synthesis in colorectal cancer patients.

FIG. 6 graphically depicts plasma glucose levels of patients receivingeither the specific nutritional combination (SNC) or the controlproduct.

DESCRIPTION OF THE INVENTION

It is an object of the invention to provide a composition suitable forimproving the muscle function, preferably leading to an improvement indaily activities of a mammal. In particular, it is an object of thepresent invention to provide a nutritional composition for such purpose.

It has now been found that it is possible to treat a subject with areduced muscle function or being at risk of developing a reduced musclefunction with a specific composition comprising proteinaceous matter.

Accordingly, the present invention relates to a combination of wheyprotein, leucine and at least one at least a ω-3 polyunsaturated fattyacid selected from the group of eicosapentaenoic acid, docosahexaenoicacid, eicosatetraenoic acid and docosapentaenoic acid for improving themuscle function in a mammal.

The invention further relates to the a combination of whey protein,leucine and at least one at least a ω-3 polyunsaturated fatty acidselected from the group of eicosapentaenoic acid, docosahexaenoic acid,eicosatetraenoic acid and docosapentaenoic acid for a use selected fromthe group of improving daily activity, improving physical performance,providing a better prognosis in terms of extended life-expectancy,improving compliance to an anti-cancer therapy and improving a qualityof life. This use may be in addition to improving the muscle function ina mammal or independent thereof.

Such a combination may in particular be in the form of a specificnutritional composition.

Accordingly, the present invention in particular relates to anutritional composition, comprising

-   -   (a) at least 18 en % of proteinaceous matter, said proteinaceous        matter comprising whey;    -   (b) at least 12 wt % of leucine, based on total proteinaceous        matter; and    -   (c) a lipid fraction comprising at least a ω-3 polyunsaturated        fatty acid selected from the group of eicosapentaenoic acid,        docosahexaenoic acid, eicosatetraenoic acid and docosapentaenoic        acid.

In particular such composition may be used for improving the musclefunction in a mammal.

Alternatively or in addition, the composition may in particular be usedfor a use selected from the group of improving daily activity, improvingphysical performance, providing a better prognosis in terms of extendedlife-expectancy, improving compliance to an anti-cancer therapy andimproving a quality of life.

When referring to a composition comprising whey protein (or in short:whey), leucine and at least one of said ω-3 polyunsaturated fatty acidsfor a specific purpose—such as for improving the muscle function in amammal—it is in particular meant these said components are intended tobe used for that purpose in combination. Accordingly, each of saidcomponents is considered to play a role in accomplishing that purpose.

The energetic value of a compound (en %) is based on the energy providedby the digestible part (in particular in a human) of the compound. Inparticular, the energetic value is based on the contribution ofproteinaceous matter, lipids and digestible carbohydrates, using thefollowing calculation factors: 4 kcal/g for digestible carbohydrates andproteinaceous matter and 9 kcal/g for lipids.

Preferably, the organoleptic properties of the composition are such thatthe consumption is generally appreciated as pleasant.

Preferably, the composition passes the stomach easily.

Preferably, the digestible components of the composition become readilyavailable upon intake of the product.

A composition according to the invention may in particular be used toimprove skeletal muscle function in a mammal. It is understood thatimproving skeletal muscle function may comprise improving either one orboth of muscle mass dependent and muscle mass independent loss of musclefunction in a mammal. For muscle mass dependent muscle function loss, itmay comprise a correction in maximal force, maximal contraction velocityor maximal relaxation velocity of skeletal muscle. For muscle massindependent loss of muscle function, it may comprise a correction inmaximal force corrected for muscle mass, maximal contraction velocitycorrected for muscle mass or maximal relaxation velocity corrected formuscle mass. Furthermore, the time needed for a contraction orrelaxation might be improved.

In an embodiment, a composition of the invention may be used to preventor treat a reduction of muscle function due to, or resulting from aging,disease, disorder, drugs or trauma, preferably drug, disease ordisorder.

Reduced muscle function may in particular manifest itself as a symptomdue to a disease or disorder such as cancer, HIV infection, COPD, renalfailure, heart failure, and a disease state characterized by a highplasma and/or serum level of pro-inflammatory cytokines. Hence, thecomposition of the invention may in particular be used for treating amammal suffering from a disease or disorder selected from the group ofcancer, HIV infection, COPD, renal failure, heart failure, and a diseasestate characterized by a high plasma and/or serum level ofpro-inflammatory cytokines.

Preferably, the disease or disorder is a cancer. In this context thereduced muscle function may be a muscle mass dependent function loss ora muscle mass independent function loss.

Moreover, treatment with drugs such as chemotherapy might lead todecrease in muscle function. Therefore, the invention relates also to anutritional composition according to the invention, wherein the drug isadministered in the framework of a chemotherapy.

Based on experiments wherein compositions according to the invention arefed to tumour-bearing mice, as illustrated in the example below, it iscontemplated by the inventors that a composition of the invention iseffective in improving the muscle function of a mammal. It was shown inthe experiments that at least one of several physiological parametersassociated with a reduced muscle function was positively affected.

It is further contemplated that a composition of the invention may beused to provide a better prognosis in terms of extended life-expectancyand/or a better quality of life. Factors improving the quality of lifeare in particular less fatigue, improved daily activity, more stamina,improved contrast between day time and night time activity (nocturnalsleep), better general condition and less periods of feeling depressed.

Proteinaceous Matter

Proteinaceous matter is formed by moieties formed from amino acids. Theterm amino acids as used herein includes amino-acid residues (e.g. inpeptides). In particular, the term ‘proteinaceous matter’ includes freeamino acids, amino acid salts, amino acid esters, the amino acidresidues bound to conjugating molecules and peptides, includingproteins. Likewise, when reference is made to a specific amino acid,e.g. leucine, this is meant to include the specific amino acid(residues) present as a salt, in a bound form, as well as the freespecific amino acid.

With a peptide is meant a combination of two or more amino acids,connected via one or more peptidic bonds. When incorporated in apeptide, amino acids are named amino-acid residues. Peptides includeoligopeptides and polypeptides, including proteins.

With a polypeptide is meant a peptide chain comprising 14 or moreamino-acid residues. With an oligopeptide is meant a peptide chaincomprising 2 to 13 amino-acid residues.

Chiral amino acids present in a composition of the invention may be inthe L-form or the D-form. Usually, the chiral amino acids present in acomposition of the invention are in the L-form.

In an embodiment, a liquid composition according to the inventioncomprises at least 7 g/100 ml of proteinaceous matter, preferably atleast 8 g/100 ml, more preferably at least 9 g/100 ml, most preferablyat least 10 g/100 ml.

The proteinaceous matter in a composition of the invention provides atleast 18 en %, preferably at least 20 en %, more preferably at least 22en % of the total composition. The proteinaceous matter in a compositionof the invention usually provides 60 en % or less, preferably 40 en % orless, or more preferably 32 en % or less of the total composition.

The proteinaceous matter comprises whey protein. Whey protein is ininter alia considered advantageous, because it has a fast post-prandialrelease of amino acids into blood, compared to e.g. casein. Thus, theinventors realised the threshold concentration of amino acids needed toswitch on the anabolic signal of muscle protein synthesis can be reachedeasier (e.g. at a lower dosage of proteinaceous matter, or sooner afteringestion).

The proteinaceous matter may further comprises proteinaceous matter fromone or more other protein sources, in particular one or moreproteinaceaous matter sources selected from the group of casein,caseinate, soy and wheat, preferably casein. Said protein source or partthereof may have been modified, in particular by (partial) hydrolysis.

With whey is meant a source of a globular protein that can be isolatedfrom whey. In particular, globular whey proteins can be selected frombeta-lactoglobulin, alpha-lactalbumin and serum albumin, includingmixtures thereof. Examples of mixtures that contain whey proteins arewhey isolate and whey concentrate. Both sources contain predominantlyintact whey proteins, which is preferred in the contect of thisapplication.

In an embodiment, the proteinaceous matter comprises at least 10 wt %,preferably at least 15 wt %, more preferably at least 20 wt %, mostpreferably at least 25 wt % of whey, based on the total proteinaceousmatter. Usually, the whey fraction is 50 wt. % or less based on totalproteinaceous matter, in particular 40 wt % or less based on totalproteinaceous matter, although—if desired—more than 50 wt. % to 100% ofthe proteinaceous matter may be provided by whey.

In particular, in case of a liquid composition, the concentration ofdenaturated whey preferably does not exceed 35 wt % based on totalproteinaceous matter. This is advantageous with respect to avoiding therisk of gelation during storage. Also, the choice of whey over freeamino acids is preferred as free amino acids have a bad taste.

The presence of whey may offer a number of advantages. The whey shows anadvantageous release behaviour both in terms of release rate of theamino acids and the tendency to make the amino acids available foruptake by the body, essentially at the same time.

The advantageous amino-acid release behaviour may be further enhanced by(slightly) hydrolysing at least part of the whey protein, usually to theextent that up to 20% of the protein is hydrolysed to free amino acids,preferably to the extent that up to 10% of the protein is hydrolysed tofree amino acids.

For said enhanced effect usually 50 wt % of the whey protein or less is(slightly) hydrolysed, in particular 10 to 50 wt %.

If desired the free amino acid or part thereof may be removed from thehydrolysate. Suitable techniques are known, e.g. filtration,chromatography or absorption.

As the source for whey protein(s) preferably a whey fraction is chosencomprising less that 20 wt % casein glycomacropeptide (GMP), morepreferably less than 10 wt %.

The beta-lactoglobulin content preferably is larger than 40 wt %, morepreferably 46 to 80 wt %.

When used as intact protein, the casein preferably comprises a highconcentration of beta casein, in particular more than 36 g/100 g casein,more in particular 38 to 70 g/100 g casein.

In an embodiment, at least part of the proteinaceous matter is presentin the form of free amino acids, a salt thereof or as a conjugate with aconjugating molecule other than a protein or peptide, which conjugate iscapable of being split in the free amino acid (or salt thereof) and theconjugating compound under the influence of a bile constituent and/or apancreas excretes in duodenum and/or the ileum. In an embodiment, theamount of amino acid in such form, in particular in the form of a saltor the free form, is up to 15 wt % based on total proteinaceous matter,preferably 0.5-14 wt %.

The peptide content (oligopeptide, polypeptide, protein) based on totalproteinaceous matter is usually at least 50 wt %, at least 60 wt % or atleast 75 wt %. The wt % of peptide based on total proteinaceous matteris usually up to 99 wt %, preferably up to 94 wt %, more preferably 89wt %.

An advantage of a composition wherein the peptide content is high (≧50wt %) is that the taste, or another organoleptic property of thecomposition, usually is appreciated better when consumed (orally).Further, the uptake of amino acids by the body may be more gradual.

In a particular embodiment, the composition comprises leucine in theform of a free acid, a salt, a dipeptide or a conjugate with aconjugating compound other than an amino acid, a protein, or a peptide,which conjugate is capable of being split into the free amino acid (orsalt thereof), preferably in the gut or stomach or after absorption inthe enterocytes or liver.

Leucine is preferably for at least 35 wt %, more preferably for at least40 wt %, based on the total proteinaceous leucine, present in the formof a peptide (oligopeptide, polypeptide, protein), preferably in theform of polypeptides and/or (intact) proteins.

Leucine is for up to 100 wt %, preferably for up to 80 wt %, based onthe total proteinaceous leucine, present in the form of a peptide(oligopeptide, polypeptide, protein), more preferably in the form of oneor more polypeptides and/or one or more (intact) proteins.

The leucine content in a composition of the invention is at least 12 wt%, at least 13 wt %, at least 16 wt % or at least 19 wt %, based ontotal proteinaceous matter. Usually the leucine content is 50 wt % orless, In particular, it may be 30 wt % or less, 25 wt % or less or 23 wt% or less, based on total proteinaceous matter. In an embodiment, theleucine content is 12 to 23 wt %, based on total proteinaceous matter.

Advantageously, the composition may comprise glutamine and/or glutamicacid.

If present, the glutamine content (determined as total glutamine &glutamic acid) is at least 15 wt %, based on total proteinaceous matter.In an embodiment, the glutamine content is 16 to 28 wt %, preferably 17to 26 wt %, based on total proteinaceous matter.

Advantageously, the composition may comprise one or more of the group ofcystine, cysteine and cysteine equivalents such as N-acetyl cysteine.preferably in an amount of at least 0.8 wt %, based on totalproteinaceous matter. Usually the content of cystine, cysteine andcysteine equivalents is 11 wt % or less, In particular, it is 8 wt % orless, based on total proteinaceous matter. In an embodiment, the contentof cystine, cysteine and cysteine equivalents is 0.8 to 8 wt %, based ontotal proteinaceous matter.

Glutathione homeostasis plays a role in maintaining whole bodyresistance to oxidative stress. Severe oxidative stress in the musclemight lead to decreased muscle function. In experiments withtumour-bearing mice, the inventors found that glutathione levels of theliver were significantly decreased. The liver is the main distributor ofglutathione and therefore liver glutathione is a good reflection ofwhole body glutathione. Surprisingly, further experiments by theinventors revealed that at least partial normalization of theglutathione level in liver cells occurred under the influence ofglutamine and/or cysteine in a composition of the invention.Particularly good results were obtained when both amino acids werepresent in the composition. Based on these experiments, it iscontemplated by the inventors that a composition of the inventioncomprising glutamine or cysteine, preferably in a concentration asindicated above, is particularly effective in improving the musclefunction of a mammal. It is further contemplated that the presence ofboth glutamine and cysteine in a composition of the invention is evenmore effective in improving the muscle function of a mammal.

In an embodiment, an advantageous effect of glutamine and/or cysteine onthe muscle function of a mammal is obtained with a composition of theinvention comprising whey protein and casein.

In a composition according to the invention the weight ratioleucine/(valine+isoleucine) is generally 1.0 or more, preferably 1.05 ormore.

In the total product the content of essential amino acids usually is atleast 49 wt %, preferably 49 to 80 wt %, more preferably 52 to 70 wt %of the total proteinaceous matter is formed by essential amino acids.

The lysine content usually is 7 to 15 g/100 g of proteinaceous matter,preferably 7.5 to 14 g/100 g of proteinaceous matter.

Decreasing muscle protein breakdown with the composition of theinvention may also help reduce carnitine and/or lysine loss fromcatabolic muscle and help maintain muscle carnitine and lysine levels.In this way, the composition of the invention comprising carnitine mightaid to skeletal muscle function. L-carnitine (beta-acetoxy-gamma-N, N,N-trimethylaminobutyrate) is synthesized from the essential aminoacidslysine and methionine mainly in liver and kidney. Carnitine is requiredfor the transport of medium/long-chain fatty acids across mitochondrialmembranes, which then can enter beta-oxidation. In addition, itfacilitates the removal of short chain organic acids from mitochondria,thereby freeing intramitochondrial coenzyme-A to participate in thebeta-oxidation and Krebscyscle. Because of these key functions,carnitine is concentrated in tissues that use fatty acids as a primarydietary fuel, such as skeletal and heart muscle.

Carnitine deficiency has been reported in several forms of cancer andhas been associated with increased fatigue. Three open labels studiesindeed suggest that treatment with carnitine reduced fatigue, measuredwith a fatigue score. Especially one derivative of carnitine,acid-soluble acylcarnitine seems to be decreased in cancer patientscompared to healthy controls. For total carnitine, a significantdecrease has been reported after three months of therapy and seems tosuggest that carnitine deficiency is induced by chemotherapy. Anotherexplanation for the onset of a carnitine deficiency may be that thedecrease in carnitine levels is associated with the presence ofcachexia. In conclusion, carnitine deficiency seems to be induced byboth chemotherapy and progression of disease (cachexia). These findingssuggest that supplementation with a composition of the inventioncomprising carnitine may best start immediately after diagnosis in orderto prevent a deficiency.

If present, the carnitine content in a composition of the invention isusually at least 5 mg per 100 kcal, preferably at least 10 mg per 100kcal, at least 25 mg per 100 kcal or at least 100 mg per 100 kcal.Usually the carnitine content is 2.5 g or less per 100 kcal, inparticular 1.25 g or less per 100 kcal. In case of a liquid product, thecarnitine content is preferably at least 10 mg/100 ml, at least 50mg/100 ml or at least 200 mg/100 ml. Usually the carnitine content is 5g or less per 100 ml, in particular 2.5 g or less per 100 ml.

Taurine is the most abundant free amino acid in cardiac and skeletalmuscle and with decrease of muscle mass it is excreted from the muscle.Taurine is thought to play an important role in ion movement and calciumhandling of the muscle and might therefore influence muscle performance.Taurine depletion causes cardiomyocyte atrophy, mitochondrial andmyofiber damage and cardiac dysfunction, effects likely related to theactions of taurine. Decreasing muscle protein breakdown using acomposition of the invention may also help reduce taurine loss fromcatabolic muscle and help maintain muscle taurine levels and in this waymaintain muscle function.

If present, the taurine content in a composition of the invention isusually at least 5 mg per 100 kcal, preferably at least 10 mg per 100kcal, at least 25 mg per 100 kcal or at least 100 mg per 100 kcal.Usually the taurine content is 2.5 g or less per 100 kcal, in particular1.25 g or less per 100 kcal. In case of a liquid product, the taurinecontent is preferably at least 10 mg/100 ml, at least 50 mg/100 ml or atleast 200 mg/100 ml. Usually the taurine content is 5 g or less per 100ml, in particular 2.5 g or less per 100 ml.

Lipid Fraction

In a composition of the invention, the lipid fraction usually providesat least 10 en %, preferably at least 20 en % or more preferably atleast 25 en % of the total composition. The lipid fraction in acomposition of the invention usually provides 50 en % or less,preferably 40 en % or less, or more preferably 35 en % or less of thetotal composition.

With the term ‘lipid fraction’ is meant a fraction comprising one ormore lipids, including fatty acids, fatty-acid derivatives (includingtri-, di-, and monoglycerides and phospholipids) and sterol-containingmetabolites such as cholesterol.

As indicated above, a composition of the invention comprises at leastone ω-3 polyunsaturated fatty acid selected from the group ofeicosapentaenoic acid (EPA), docosahexaenoic acid (DHA),eicosatetraenoic acid (ETA) and docosapentaenoic acid (DPA).

A composition of the invention may further comprise ω-3 and/or ω-6polyunsaturated fatty acids, in particular those containing 18 to 26carbon atoms, e.g. linolenic acid (LA), alpha linolenic acid (ALA),gamma linolenic acid (GLA), dihomo gamma-linolenic acid (DGLA) andarachidonic acid (AA).

For obtaining an advantageous effect on muscle function, the ω-3unsaturated fatty acid content is usually at least 10 wt %, preferablyat least 15 wt %, based on total lipid content.

In a further embodiment, the composition of the invention comprisesstearidonic acid (SDA). Nutritional oils containing SDA are reported tobe a dietary source of ω-3 fatty acids that would be more effective inincreasing tissue EPA and DPA concentrations than are currentALA-containing oils. Preferably, the lipid fraction in the compositioncomprises more than 0.5 wt % of SDA, more preferable more than 0.6 wt %of SDA, still more preferably more than 1.2 wt % of SDA, based on totallipid. The maximum amount is more or less limited by the particularsource used (type of marine oil), but marine oils with an SDA amount of2 wt % to about 5 wt % (based on total lipid in the latter oil) arecommercially available. Preferably, the amount of SDA in the lipidfraction ranges between 0.5 and 5 wt %, based on total lipid. It ispreferred that the amount of SDA is relatively high compared to that ofdocosahexaenoic acid (DHA) and/or linoleic acid (LA). This allows a highefficacy and manufacture of palatable products comprising low amounts ofoxidized products. In effective embodiments of the product according theinvention the weight ratio of SDA to DHA is therefore at least 0.22,preferably at least 0.25, more preferably at least 0.30.

A composition of the invention may in particular be a compositionwherein at least 55 wt % of the lipid fraction, preferably triglycerideoils, comprise at least 4 wt % of one or more of eicosapentaenoic acidand docosahexaenoic acid.

In a composition of the invention, the lipid fraction comprises lessthan 30 wt % of a saturated fatty acid, preferably less than 22 wt %,based on total lipid content.

The ratio ω-3 to ω-6 polyunsaturated fatty acids can be chosen withinwide limits, e.g. from 0.2 to 10, or from 0.4 to 3.0. In particular, theratio ω-3 to ω-6 polyunsaturated fatty acids is less than 1.0,preferably 0.97 or less, more preferably 0.95 or less. The ratio ispreferably larger 0.5 or more, more preferably 0.6 or more. Inparticular, preferably the ratio is from 0.5 to 0.97, more preferablyfrom 0.6 to 0.95.

Carbohydrate Fraction

In an embodiment, a composition of the invention comprises a digestiblecarbohydrate fraction, providing at least 20 en %, preferably at least30 en % or more preferably at least 38 en % of the total composition.

The digestible carbohydrate fraction in a composition of the inventionusually provides 70 en % or less, preferably 60 en % or less, morepreferably 48 en % of the total composition.

With the term ‘digestible carbohydrate’ fraction is meant a fractioncomprising one or more digestible carbohydrates.

Digestible carbohydrates include maltodextrose, maltose and glucose. Inparticular, a carbohydrate is considered digestible in case more than90% of quickly carbohydrates are digested within 20 min in accordancewith the Enquist method.

Especially the composition of the carbohydrate fraction may be chosen toachieve a favourable carbohydrate uptake, and accordingly a desirableinsulin release after intake. Accordingly, in particular a compositionmeeting one or more of the following criteria with respect to thecarbohydrate content are considered to be advantageous.

In an embodiment less than 75 wt % of the carbohydrates is formed by thesum of the sucrose and the maltodextrin content.

In an embodiment at least 40 wt % based on the total weight of thecarbohydrates is formed by slowly digestible carbohydrates, i.e. inparticular carbohydrates which are digested less fast thanmaltodextrose, maltose and glucose

In an embodiment a composition according to the invention comprises lessthan 60 wt %, preferably 20 to 50 wt % based on the total weight of thecarbohydrates of quickly digestible carbohydrates, in particular ofmaltodextrose, maltose, glucose and other carbohydrates which aredigested at least as fast.

In an embodiment more than 20 wt % based on the total weight of thecarbohydrates is formed by at least one disaccharide, preferably 22 to60 wt %. In particular, in such an embodiment, the disaccharide ispreferably selected from the group consisting of sucrose, trehalose,palatinose, lactose and other low glycemic disaccharides, morepreferably from trehalose and palatinose.

In an embodiment at least one monosaccharide other than glucose ispresent. Preferably said monosaccharide is selected from the groupconsisting of galactose, mannose and ribose. Preferably the total amountof said monosaccharide(s) is 0.5 to 30 wt %, more preferably 5 to 25 wt% based on the total weight of the carbohydrates.

In particular, the presence of ribose is advantageous, preferably incombination with (endogenous) folic acid, to increase the proteinsynthesis. It is contemplated that the combination of these twocompounds allows an increase in the production of guanosine triphosphatein the mammal, resulting in an increase of the protein synthesis viastimulation of eukaryotic initiation factor 2B, especially inmalnourished patients. The folic acid may be provided in one or more ofthe following forms: free folic acid, folinic acid, formylated folicacid, methylated folic acid, preferably in a reduced form or as a mono-or polyglutamate conjugated derivative. When present, the folic acidcontent is usually at least 95 μg per 100 kcal carbohydrates, preferably110 to 400 μg per 100 kcal carbohydrates, more preferably 125 to 300 μgper 100 kcal carbohydrates.

It is contemplated that it is advantageous with respect to improving themuscle function in a mammal, improving daily activity, improvingphysical performance, providing a better prognosis in terms of extendedlife-expectancy, improving compliance to an anti-cancer therapy or withrespect to improving a quality of life, that the composition has arelatively low glycemic index. Without being bound by theory it iscontemplated that a combination or composition according to theinvention to having a low glycemic index is advantageous with respect tomuscle protein synthesis, and/or muscle strength, because it contributesto a high insulin sensitivity of the muscle. A high insulin sensitivityis considered to be beneficial in that it improves the stimulatingeffect of insulin on muscle synthesis (insulin stimulation being atrigger for switching on the anabolic signal of muscle).

Accordingly, in a specific embodiment, the composition is a nutritionalcomposition with a low glycemic index. In particular it is consideredadvantageous that the glycemic index of the composition is below 55,preferably below 45. In practice, the glycemic index will be above zero,and usually be at least 1, in particular at least 5. Details on how todetermine the glycemic index of a composition are provided in theExamples, herein below.

The skilled person will be able to formulate a composition with arelatively low glycemic index based on the information disclosed hereinand common general knowledge. In particular, by increasing thepercentage of carbohydrate that is digested more slowly than glucose orby increasing carbohydrates that provide less glucose moieties perweight than glucose, the glycemic index of a composition (underotherwise the same condition) is decreased. Preferred examples ofcarbohydrates which are digested more slowly than glucose areisomaltulose, fructose, galactose, lactose, trehalose. Next to thataddition of fat and fibre can slow down gastric emptying. Moreover,fibres can form a physical barrier in the intestine, reducing absorptionrate. Amino acids from protein can increase insulin release (especiallyleucine), and thereby increase glucose uptake by the cells. All thesemechanisms can contribute to a reduction in glycemic index.

Indigestible Carbohydrate Fraction

In an embodiment, a composition of the invention comprises anindigestible carbohydrate fraction,

In a preferred composition, the indigestible carbohydrate is selectedfrom the group of galactooligosaccharides and fructooligosaccharides.

In particular, the galactooligosaccharide is selected from the group ofshort-chain galactooligosaccharides, long-chain galactooligosaccharides,or any combination thereof.

In particular, the fructooligosaccharide is selected from the group ofshort-chain fructooligosaccharides, long-chain fructooligosaccharides,or any combination thereof.

A preferred composition comprises a galactooligosaccharide and afructooligosaccharide.

Preferably, the molar ratio of galactooligosaccharide tofructooligosaccharide ranges from 1:1 to 20:1, preferably from 5:1 to12:1, and is most preferably equal to about 9:1.

With an oligosaccharide is meant a chain comprising 2 to 25 saccharideresidues.

With a long chain oligosaccharide is meant an oligosaccharide chaincomprising 10-25 saccharide residues. With a short chain oligosaccharideis meant an oligosaccharide chain comprising 2-9 saccharide residues,for example 2-5 residues or 6-9 residues.

Indigestible carbohydrates are carbohydrates that remain in essenceundigested in the human intestines. In particular, a carbohydrate isconsidered indigestible in case less than 10% of the sugars is releasedwithin 20 and 120 min in an analysis setting using standard digestiveenzymes, as determinable by the Enquist method.

In a particular embodiment, the indigestible carbohydrate is selectedfrom the group of galactomannans having a degree of polymerisation (DP)between 2 and 50, xylans with a DP of 2 to 60, oligomers having morethan 30 wt % of galacturonic acid or glucuronic acid moieties having amolecular weight of 520 to 2200 Dalton, and any combination thereof.

In an embodiment, the indigestible carbohydrate content is at least 1 wt%, at least 2 wt % or at least 3 wt %, based on total dry matter. In anembodiment, the indigestible carbohydrate content amounts 1 to 15 wt %,preferably 2 to 12 wt %, more preferably 3 to 10 wt %, based on totaldry matter.

In a specific embodiment of the present invention, the compositionaccording to the invention may comprise a mixture of neutral and acidoligosaccharides as disclosed in WO 2005/039597 (N.V. Nutricia), whichis incorporated herein by reference in its entirety. More in particular,the acid oligosaccharide has a degree of polymerization (DP) between 2and 5000, preferably between 2 and 1000, more preferably between 2 and250, even more preferably between 2 and 50, most preferably between 2and 10. If a mixture of acid oligosaccharides with different degrees ofpolymerization is used, the average DP of the acid oligosaccharidemixture is preferably between 2 and 1000, more preferably between 3 and250, even more preferably between 3 and 50. The acid oligosaccharide maybe a homogeneous or heterogeneous carbohydrate. The acidoligosaccharides may be prepared from pectin, pectate, alginate,chondroitine, hyaluronic acids, heparin, heparane, bacterialcarbohydrates, sialoglycans, fucoidan, fucooligosaccharides orcarrageenan, and are preferably prepared from pectin or alginate. Theacid oligosaccharides may be prepared by the methods described in WO01/60378, which is hereby incorporated by reference.

The acid oligosaccharide is preferably prepared from high methoxylatedpectin, which is characterized by a degree of methoxylation above 50%.As used herein, “degree of methoxylation” (also referred to as DE or“degree of esterification”) is intended to mean the extent to which freecarboxylic acid groups contained in the polygalacturonic acid chain havebeen esterified (e.g. by methylation). The acid oligosaccharides arepreferably characterized by a degree of methoxylation above 20%,preferably above 50% even more preferably above 70%. Preferably the acidoligosaccharides have a degree of methylation above 20%, preferablyabove 50% even more preferably above 70%. The acid oligosaccharide ispreferably administered in an amount of between 10 mg and 100 gram perday, preferably between 100 mg and 50 grams per day, even more between0.5 and 20 gram per day.

The term neutral oligosaccharides as used in the present inventionrefers to saccharides which have a degree of polymerization of monoseunits exceeding 2, more preferably exceeding 3, even more preferablyexceeding 4, most preferably exceeding 10, which are not or onlypartially digested in the intestine by the action of acids or digestiveenzymes present in the human upper digestive tract (small intestine andstomach) but which are fermented by the human intestinal flora andpreferably lack acidic groups. The neutral oligosaccharide isstructurally (chemically) different from the acid oligosaccharide. Theterm neutral oligosaccharides as used in the present inventionpreferably refers to saccharides which have a degree of polymerizationof the oligosaccharide below 60 monose units, preferably below 40, evenmore preferably below 20, most preferably below 10. The term monoseunits refers to units having a closed ring structure, preferably hexose,e.g. the pyranose or furanose forms. The neutral oligosaccharidepreferably comprises at least 90%, more preferably at least 95% monoseunits selected from the group consisting of mannose, arabinose,fructose, fucose, rhamnose, galactose, □-D-galactopyranose, ribose,glucose, xylose and derivatives thereof, calculated on the total numberof monose units contained therein. Suitable neutral oligosaccharides arepreferably fermented by the gut flora. Preferably the oligosaccharide isselected from the group consisting of: cellobiose(4-O-β-D-glucopyranosyl-D-glucose), cellodextrins((4-O-β-D-glucopyranosyl)_(n)-D-glucose), B-cyclodextrins (Cyclicmolecules of α-1-4-linked D-glucose; α-cyclodextrin-hexamer,β-cyclodextrin-heptamer and γ-cyclodextrin-octamer), indigestibledextrin, gentiooligosaccharides (mixture of β-1-6 linked glucoseresidues, some 1-4 linkages), glucooligosaccharides (mixture ofα-D-glucose), isomaltooligosaccharides (linear α-1-6 linked glucoseresidues with some 1-4 linkages), isomaltose(6-O-α-D-glucopyranosyl-D-glucose); isomaltriose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-D-glucose), panose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-(1-4)-D-glucose),leucrose (5-O-α-D-glucopyranosyl-D-fructopyranoside), palatinose orisomaltulose (6-O-α-D-glucopyranosyl-D-fructose), theanderose(O-α-D-glucopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-B-D-fructofuranoside),D-agatose, D-lyxo-hexulose, lactosucrose(O-β-D-galactopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside),α-galactooligosaccharides including raffinose, stachyose and other soyoligosaccharides(O-α-D-galactopyranosyl-(1-6)-α-D-glucopyranosyl-β-D-fructofuranoside),β-galactooligosaccharides or transgalacto-oligosaccharides(β-D-galactopyranosyl-(1-6)-[P-D-glucopyranosyl]_(n)-(1-4) α-D glucose),lactulose (4-O-β-D-galactopyranosyl-D-fructose), 4′-galatosyllactose(O-D-galactopyranosyl-(1-4)-O-β-D-glucopyranosyl-(1-4)-D-glucopyranose),synthetic galactooligosaccharide (neogalactobiose, isogalactobiose,galsucrose, isolactose I, II and III), fructans—Levan-type(β-D-(2→6)-fructofuranosyl)_(n) α-D-glucopyranoside),fructans—Inulin-type (β-D-((2→1)-fructofuranosyl)_(n)α-D-glucopyranoside), 1 f-β-fructofuranosylnystose(β-D-((2→1)-fructofuranosyl)_(n) B-D-fructofuranoside),xylooligosaccharides (B-D-((1→4)-xylose)_(n), lafinose, lactosucrose andarabinooligosaccharides.

According to a further preferred embodiment the neutral oligosaccharideis selected from the group consisting of fructans,fructooligosaccharides, indigestible dextrins galactooligosaccharides(including transgalactooligosaccharides), xylooligosaccharides,arabinooligosaccharides, glucooligosaccharides, mannooligosaccharides,fucooligosaccharides and mixtures thereof. Most preferably, the neutraloligosaccharide is selected from the group consisting offructooligosacchararides, galactooligosaccharides andtransgalactooligosaccharides.

Suitable oligosaccharides and their production methods are furtherdescribed in Laere K. J. M. (Laere, K. J. M., Degradation ofstructurally different non-digestible oligosaccharides by intestinalbacteria: glycosylhydrolases of Bi. adolescentis. PhD-thesis (2000),Wageningen Agricultural University, Wageningen, The Netherlands), theentire content of which is hereby incorporated by reference.Transgalactooligosaccharides (TOS) are for example sold under thetrademark Vivinal™ (Borculo Domo Ingredients, Netherlands). Indigestibledextrin, which may be produced by pyrolysis of corn starch, comprisesα(1→4) and α(1→6) glucosidic bonds, as are present in the native starch,and contains 1→2 and 1→3 linkages and levoglucosan. Due to thesestructural characteristics, indigestible dextrin containswell-developed, branched particles that are partially hydrolysed byhuman digestive enzymes. Numerous other commercial sources ofindigestible oligosaccharides are readily available and known to skilledperson. For example, transgalactooligosaccharide is available fromYakult Honsha Co., Tokyo, Japan. Soybean oligosaccharide is availablefrom Calpis Corporation distributed by Ajinomoto U.S.A. Inc., Teaneck,N.J.

In a further preferred embodiment, the composition according to theinvention comprises an acid oligosaccharide with a DP between 2 and 250,prepared from pectin, alginate, and mixtures thereof; and a neutraloligosaccharide, selected from the group of fructans,fructooligosaccharides, indigestible dextrins, galactooligosaccharidesincluding transgalactooligosaccharides, xylooligosaccharides,arabinooligosaccharides, glucooligosaccharides, mannooligosaccharides,fucooligosaccharides, and mixtures thereof.

In a further preferred embodiment the composition according to theinvention comprises two chemically distinct neutral oligosaccharides. Itwas found that the administration of acid oligosaccharides combined withtwo chemically distinct neutral oligosaccharides provides an optimalsynergistic immune stimulatory effect.

Preferably the composition according to the invention comprises:

-   -   an acid oligosaccharides as defined above;    -   a galactose-based neutral oligosaccharide (of which more than        50% of the monose units are galactose units), preferably        selected from the group consisting of galactooligosaccharide and        transgalactooligosaccharide; and    -   a fructose and/or glucose based neutral oligosaccharide (of        which more than 50% of the monose units are fructose and/or        glucose, preferably fructose units), preferably inulin, fructan        and/or fructooligosaccharide, most preferably long chain        fructooligosaccharide (with an average DP of 10 to 60).

A mixture of acid- and neutral oligosaccharides is preferablyadministered in an amount of between 10 mg and 100 gram per day,preferably between 100 mg and 25 grams per day, even more preferablybetween 0.5 and 20 gram per day.

Nutritional Composition

With a nutritional composition is meant a composition that comprisesnaturally occurring components, preferably found in the food supply,that can be sold over the counter, as supplements, functional foods orfood ingredients i.e. without a physician's or veterinarian'sprescription. A nutritional composition may also be a medical food,intended for the dietary management of a disease or condition formammals under the supervision of a physician or veterinarian.

A composition according to the invention may be in the form of a liquid,e.g. a drink, in the form of a semi-liquid, e.g. a yoghurt or a custard,in the form of a gel, e.g. jelly cake or in the form of a solid, e.g. acandy bar or an ice-cream.

In an embodiment, a liquid composition is prepared from a concentrate,e.g. from a liquid (e.g. with a viscosity of less than about 80 mPa·s),a semi-liquid (e.g. with a viscosity of more than about 80 mPa·s andless than about 400 mPa·s), a gel or a solid. For such preparation,water may be used to dilute the concentrate. In particular, suchpreparation occurs just before administration of the composition, e.g.in an instant-fashion.

One particular embodiment of the invention is a nutritional compositioncomprising proteinaceous matter, a lipid, and a digestible carbohydrate,wherein

-   -   a) the proteinaceous matter content provides 18 to 60 en %, in        particular 18 to 50 en %, preferably 20 to 40 en %, more        preferably 22 to 32 en % of the total composition, said        proteinaceous matter comprising whey;    -   b) the lipid content provides 10 to 50 en %, preferably 20 to 40        en %, more preferably 25 to 35 en % of the total composition;    -   c) the digestible carbohydrate content provides 20 to 70 en %,        preferably 30 to 60 en %, more preferably 38 to 48 en % of the        total composition.

The total energetic value of a liquid composition in accordance with theinvention may be chosen within wide limits, e.g. from 0.2 to 4 kcal/ml.Usually it is at least 0.3 kcal/ml, in particular at least 0.8 kcal/ml,more in particular at least 1.2 kcal/ml. Usually, it is 3.0 kcal/ml orless, in particular 2.6 kcal/ml or less, more in particular 2.4 kcal/mlor less. In a specific embodiment, the liquid composition in accordancewith the invention has an energetic value in the range of 0.3 to 3.0kcal/ml, preferably 0.8 to 2.6 kcal/ml, more preferably 1.2 to 2.4kcal/ml.

In another specific embodiment, the liquid composition in accordancewith the invention has an energetic value in the range of 0.2 to 1.0kcal/ml, preferably 0.4 to 0.9 kcal/ml.

Factors that play a role in determining a desirable energetic valueinclude the ease of achieving a higher en % proteinaceous matter on theone hand and a fast emptying of the stomach (increasing anabolicresponse) on the other hand.

The total energetic value of a semi-liquid, gel or solid composition inaccordance with the invention may be chosen within wide limits, e.g.from 1 to 15 kcal/g. Usually, it is at least 2.0 kcal/g, preferably atleast 2.8 kcal/g, even more preferably at least 3.2 kcal/g. Usually, itis 12 kcal/g or less, preferably 10 kcal/g or less, even more preferably8.0 kcal/g or less. In a specific embodiment, the semi-liquid, gel orsolid composition in accordance with the invention has an energeticvalue in the range of 3.2 to 8.0 kcal/g.

Additional Components

In an embodiment, the composition may comprise one or more otheradditional components such as at least one component selected from thegroup consisting of minerals, trace elements and vitamins, preferablyselected from the group consisting of sodium, potassium, chloride,fluoride, iodide, calcium, phosphorous, magnesium, vitamin A, vitaminD3, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B3, vitaminB5, vitamin B6, folic acid, vitamin B12, biotin, vitamin C, lipoic acid,zinc, iron, copper, manganese, molybdenum selenium and chromium.

Such components may be present in a concentration up to the dailyrecommended dose per daily serving.

Zinc is preferably present in a concentration of at least 2.8 mg per 100kcal carbohydrates, more preferably of 5.6 to 20 mg per 100 kcalcarbohydrates, even more preferably of 6-15 mg per 100 kcalcarbohydrates.

Sustained Release Preparation

In a preferred embodiment, the composition in accordance with theinvention further comprises a sustained release preparation effective torelease an amino acid in the duodenum and/or the ileum, said preparationcomprising at least one component selected from the group consisting ofamino acids in the form of a free acid, amino acids in the form of asalt and amino acids in the form of a conjugate with a conjugatingcompound other than a protein which conjugate is capable of being splitin the free amino acid (or salt thereof) and the conjugating compoundunder the influence of a bile constituent and/or a pancreas excrementsin duodenum and/or the ileum.

The amino acid in the sustained release form is preferably suspended ina liquid, semi-liquid or solid product.

The sustained release preparation can be made based upon conventionaltechniques. The amino acid(s) may be coated with a pH sensitive materialthat dissolves at the pH existing in the duodenum/ileum (about pH 7) butnot in the stomach (strongly acidic). Such coatings are generally knownin the art. Examples of conjugating molecules are molecules formingspecific peptides with the amino acid that are not split by pepsin, orat least not efficiently split under physiological conditions. Examplesare choline, betain, dimethylglycine and sarcosine. Other suitableconjugating molecules include phospholipids, lyso-phospholipids andglycerol.

Amino acids that are preferably present in the sustained releasepreparation are preferably selected from leucine and other essentialamino acids, in particular methionine, arginine, tryptophan,phenylalanine and lysine, of which leucine is especially preferred.

In an advantageous embodiment, a composition according to the inventionis administered in a drug regimen. In particular, the composition can beused as adjuvant of a drug, such as a drug selected from the groupconsisting of anti-cancer drugs, anti-retroviral drugs,antihypertensives, anti-thrombotics, anti-depressants and anti-diabeticdrugs. In particular, it is advantageous to use the product withmetformin or another anti-diabetic drug. These drugs in particular areconsidered to be stable in a composition according the invention and tobe very effective. Said drug may be present in the composition accordingto the invention or be administered separately.

The invention further relates to a method for improving the musclefunction of a mammal, comprising administering a nutritional compositioncomprising at least 18 en % of proteinaceous matter having a leucinecontent of at least 9.5 wt % based on total proteinaceous matter, alipid fraction comprising at least one ω-3-polyunsaturated fatty acidselected from the group of eicosapentaenoic acid (EPA), docosahexaenoicacid (DHA), eicosatetraenoic acid (ETA) and docosapentaenoic acid (DPA).

The compositions of the invention may be administered under thesupervision of a medical specialist, or may be self-administered.

The composition may be administered enterally or orally.

The mammal preferably is a human.

The invention will now be illustrated on the basis of the followingexamples.

DESCRIPTION OF THE FIGURES

FIG. 1: Differences in A) muscle Tibialis Anterior mass and B)Epididymal fat mass after different interventions

Con=mice receiving control diet A (AIN93), TB-con=tumor-bearing micereceiving control diet A (AIN93), hpr=high protein, leu=leucine, fo=fishoil. Data are means±SEM: * sign. different from TB-con (p<0.02) (k=5,α=10%) (For more details about statistics, see the Materials andMethods).

FIG. 2. Skeletal muscle function: force frequency curve (ex-vivo).

CON=mice receiving control diet B, TB-CON=tumor-bearing mice receivingcontrol diet B, TB-SNC=tumor-bearing mice receiving the specificnutritional combination. Data are means±SEM; data were significantlydifferent from TB-CON when p<0.05 (k=2, α=10%).

A: Maximal contraction force (complete curves significantly differentfrom each other p<0.01).

B. Maximal contraction velocity (complete curves significantly differentfrom each other p<0.01).

C. Maximal relaxation velocity (complete curves significantly differentfrom each other p<0.01).

D. CT90: time needed for contraction from 10 to 90% of maximal force(CON significantly different from TB-CON for range 83-176 Hz; TB-SNCsignificantly different from TB-CON for range 83-100 Hz).

FIG. 3. Skeletal muscle function during exercise (ex-vivo).

CON=mice receiving control diet B, TB-CON=tumor-bearing mice receivingcontrol diet B, TB-SNC=tumor-bearing receiving the specific nutritionalcombination. Data as means+/−SEM, Data were significantly different fromTB-CON when p<0.05, k=2, α=10%)

A: Maximal contraction force (both curves significant different fromTB-CON till repeat 70).

B: Maximal contraction force corrected for muscle mass (CON significantdifferent from TB-CON for repeats 30-50; TB-SNC not significantdifferent from TB-CON).

C: Maximal contraction velocity (both curves significant different fromTB-CON till repeat 70).

D: Maximal contraction velocity corrected for muscle mass (CONsignificantly different from TB-CON for the first 30 repeats (except forrepeat 5 (p=0.06)); TB-SNC significantly different from TB-CON for thefirst 10 repeats).

FIG. 4. Total daily activity.

A: Total daily activity as % of daily activity on day 2 for all groups.A significant time×group interaction was observed (P<0.01).

B: Total activity in the dark as % of daily activity on day 2 for allgroups.

C: Total activity in the light as % of daily activity on day 2 for allgroups.

A-C: * P<0.05 vs TB-CON

D: Actogram, representing percentages of daily activity during the lightperiod from 7-19 h (white shaded areas) and during the dark period from19-7 h (grey shaded areas) on days 1-19 (vertical) for all groupsseparately.

FIG. 5. Muscle protein synthesis in colorectal cancer patients.

Synthesis is expressed as fractional synthetic rate in stage IVcolorectal cancer patients receiving either a specific nutritionalcombination (SNC) or a control nutritional supplement. Fractionalsynthetic rate was measured at baseline (basal) and aftersupplementation.

FIG. 6. Plasma glucose levels of patients receiving either the specificnutritional combination (SNC) or the control product.

Examples Example 1 Materials and Methods Animals.

Male CD2F1 mice at 6-7 weeks of age, (BALB/c×DBA/2, Harlan/Charles Riverthe Netherlands) were individually housed in a climate-controlled room(12:12 dark-light cycle with a constant room temperature of 21±1° C.).After acclimatization for one week mice were divided into weight-matchedgroups: (1) control receiving control chow, (2) tumor-bearing receivingcontrol chow, and (3) tumor-bearing receiving experimental diets. Datashown are derived from the combination of several experimental runs withidentical animal characteristics and experimental procedures (unlessstated otherwise) and differ only in the experimental diets used. Allexperimental procedures were approved by the Animal Ethical Committee(DEC consult, Bilthoven, The Netherlands) and complied with theprinciples of good laboratory animal care.

Experimental Diets (Categories A and B Experiments).

Experiments are divided in: (A) experiments designed to test the effectof single or combinations of nutritional components (addition of highprotein (hpr), leucine (leu), fish oil (fo)), added to the backgrounddiet (AIN93-M) and supplied as pellets; (B) experiments designed to testthe effect of a complex nutritional combination that resembles thecomposition of Table 3 and comprises all individual components tested in(A), and differing in macronutrient composition from AIN-93 to achieve amore humanized Western-type diet, supplied as a dough for producttechnical reasons.

The AIN93-M control diet in the category A experiments contained per kgfeed: 126 g protein (100% casein), 727 g carbohydrates and 40 g fat(100% soy oil) (Research Diet Services, Wijk bij Duurstede, theNetherlands). Experimental diets in this category were adjusted tocontrol diets by partly replacing the carbohydrates and/or soy oil byprotein and leucine (151 g casein/kg and 16 g leucine/kg feed;TB+hpr+leu), high protein and fish oil (151 g casein/kg and 22 g fishoil/kg feed; TB+hpr+fo), or high protein and leucine and fish oil (151 gcasein/kg, 15 g leucine/kg and 22 g fish oil/kg food; TB+hpr+leu+fo).The 22 g fish oil contained 6.9 g EPA and 3.1 g DHA resulting in a ratioof 2.2:1.

In the category B experiment, the control diet was iso-caloric andiso-nitrogenous to the control diet in the A-category of experiments andcontained per kg feed 126 g protein (casein), 53 g fat (corn oil), and699 g carbohydrates. The iso-caloric experimental diet (further referredto as Specific Nutritional Composition; SNC) contained per kg feed: 210g protein (189 g intact protein of which 68% casein and 32% whey and 21g free leucine), 53 g fat (20.1 g corn oil, 10.2 g canola oil, and 22.2g fish oil), 561 g carbohydrates, 18 g galacto-oligosaccharides and 2 gfructooligosaccharides.

Tumor Model.

Murine C-26 adenocarcinoma cells were cultured in vitro with RPMI 1640supplemented with 5% fetal calf serum and 1% penicillin-streptomycin.Tumor cells were trypsinized in a sub-confluent state and, afterwashing, suspended in Hanks' balanced salt solution (HBSS) at aconcentration of 2.5×10⁶ cells·mL⁻¹. Under general anesthesia(isoflurane/N20/02), tumor cells (5×10⁵ cells in 0.2 mL) were inoculatedsubcutaneously into the right inguinal flank of the mice. Control (C)animals received a sham injection with 0.2 mL HBSS.

Experimental Protocol.

Following inoculation of tumor cells or HBSS, body mass, food intake andtumor size (length and width) were measured three times a week. Only inthe category B experiment, daily activity in the home cage wasmonitored. In all experiments, animals were anaesthetized and weightedat day 20 after tumor inoculation. Skeletal muscles (e.g. m. TibialisAnterior (mTA), m. Gastrocnemius (mG), m. Extensor Digitorum Longus(mEDL) and M. Soleus (mS)), the tumor, spleen, kidneys, liver,epididymal fat, thymus, lungs and heart were dissected and weighed.Carcass mass was calculated by subtracting tumor mass from body mass. Inaddition, muscle function was tested ex vivo in the category Bexperiment.

Assessment of Daily Activity.

Physical activity was monitored continuously (24 hours) during the20-day study period starting at day 2, using activity sensors (dualtechnology detector DUO 240, Visonic; adapted by R. Visser, NIN,Amsterdam, The Netherlands) that translated individual changes in theinfrared pattern caused by movements of the animals into arbitraryactivity counts. Sensors were mounted above the home cages and wereconnected via input ports and interface to a computer equipped withMED-PC IV software for data collection (MED associates, St. Albans,Vt.). Activity was expressed in counts per hour (both for the total24-hours period, the dark period (active period) and the light period(inactive period)). Activity was calculated for each mouse separatelyand was expressed relative to its own total activity on day 2, tocorrect for differences in the individual sensitivity of sensors. Theactivities of two subsequent days were averaged, to dampen the day today variability. In order to determine changes in activity patternthroughout the experiment, hourly and dark-light activity were expressedas percentage of total daily activity and translated into an actogram.

Assessment of Muscular Functionality.

Contractile characteristics of the right EDL muscle were assessed exvivo, as described previously (Gorselink, M., Vaessen, S. F., van derFlier, L. G., Leenders, I., Kegler, D., Caldenhoven, E., van der Beek,E., and van Helvoort, A. Mass-dependent decline of skeletal musclefunction in cancer cachexia. Muscle Nerve, 33: 691-693, 2006). Briefly,muscles were allowed to stabilize in the organ bath for 30 min, afterwhich optimal stimulation current and strength were determined. Thenforce-frequency characteristics (10 to 167 Hz, 250 ms) were determinedand after replenishing the organ buffer and a resting period of 5 min,muscles were subjected to an exercise protocol (83 Hz, 250 ms every 1000ms). This protocol represents a moderate load, comparable with normaldaily activity. At the frequency used, complete tetanus of the muscle isreached. Isometric force signals of the force-frequency curve wereanalyzed for maximal and total force and for maximal contraction andrelaxation velocity.

Statistics.

All data are expressed as means±SEM. Statistical analyses were performedusing SPSS 15.0 (SPSS Benelux, Gorinchem, the Netherlands). Inexperiment A different batches of animals were used, therefore, for allparameters it was defined that combination of data was allowed if nointeraction between groups and experiments were present. Bodycomposition data, tumor and organ masses on day 20 were compared betweengroups with analysis of variance (ANOVA) and post-hoc LSD. Differenceswere considered significant at a p value below α/k; in which α=10% andk=amount of comparisons. For experiment A the p-value had to be below0.02; for experiment B the p-value had to be below 0.05. Data on foodintake, body weight, daily activity, and muscle function that weremonitored during the 20 days after inoculation were analyzed by repeatedmeasures ANOVA. To further discriminate the differences between groups,the differences or deltas from the first measurement in the range werecalculated. These deltas were compared between groups using ANOVA, withpost-hoc LSD for pair-wise comparison between groups. For skeletalmuscle function, data of first measurement at day 20 were not similarbetween groups, therefore further discrimination was performed in a perpoint analysis ANOVA. Differences were considered significant at a twotailed p<0.05.

Results

Effects of Single or Combined Nutritional Components on Parameters ofCachexia.

Compared with control mice (Con), carcass and body weight weresignificantly lower in tumor bearing control mice (TB-con) on day 20after tumor inoculation (Table 1A). For all parameters measured therewas no group * experiment interaction. The loss of body weight in TB-conmice was derived from both loss of fat mass (e.g. epididymal fat) andmuscle mass (FIGS. 1A and B). No differences in food intake were presentbetween groups for complete curves. When analyzed separately per day, atday 20, Con was significantly different from TB-con. None of thetumor-bearing groups were significantly different from each other (Table1B). Addition of extra protein and leucine (TB+hpr+leu) or fish oil(TB+fo) did not change body weight compared to TB-con (Table 1).However, addition of fish oil to extra protein (TB+hpr+fo) or fish oilto extra protein and leucine (TB+hpr+leu+fo), resulted in a significanthigher fat mass compared to TB-con (FIG. 1B). Supplementation of thediet with the all-in combination of high protein, leucine and fish oil(TB+hpr+leu+fo) resulted in a significant improvement of body andcarcass weight (Table 1A), and of muscle (mTA) and fat (epididymal)mass, compared to TB-con mice (FIG. 1). Additive effects of thecombination of leucine and high protein were found for muscle mass ofthe mTA in the presence of fish oil. Addition of each componentincreased muscle mass stepwise (FIG. 1A).

TABLE 1 Effect of single or combined nutritional components on bodycomposition, and food intake. A body, tumor and carcass weightstreatment N CW p BW p TW p Con 40 24.4 ± 0.3 0.0000 * 24.4 ± 0.3 0.001 *0.0 ± 0.0 0.0000 * TB-con 40 20.7 ± 0.4 — 22.8 ± 0.4 — 2.2 ± 0.1 — TB +hpr + leu 10 20.0 ± 0.6 0.8073 21.8 ± 0.6 0.992 1.8 ± 0.1 0.1472 TB + fo10 20.9 ± 0.8 0.2264 23.0 ± 0.8 0.238 2.1 ± 0.1 0.6854 TB + fo + hpr 1022.2 ± 0.8 0.0337 24.2 ± 0.7 0.038 2.0 ± 0.1 0.5053 TB + fo + hpr + leu22 22.7 ± 0.6 0.0099 * 24.4 ± 0.5 0.019 * 1.7 ± 0.1 0.0659 B food intake(per day) treatment N 1 7 14 17 19 20 Con 40 4.5 3.8 4.0 3.8 3.7 3.6 *TB-con 40 4.2 3.9 3.8 3.8 3.5 2.9 TB + hpr + leu 10 4.7 3.8 4.0 3.1 3.72.9 TB + fo 10 5.4 3.9 4.1 4.0 3.1 2.3 TB + fo + hpr 10 4.4 3.9 3.9 3.73.3 2.6 TB + fo + hpr + leu 22 4.4 3.5 4.1 3.5 3.4 3.0 Con = micereceiving control diet A (AIN93), TB-con = tumor-bearing mice receivingcontrol diet A (AIN93), hpr = high protein, leu = leucine, fo = fishoil, CW = carcass weight, BW = body weight, and TW = tumor weight. Dataas means ± SEM: * = significantly different from TB-con (p < 0.02, k =5, α = 10%), for more details about statistics, see the Materials andMethods.

Effect of a Specific Nutritional Combination on Parameters of Cachexia.

Body and carcass weight were significantly lower in tumor-bearing mice(TB-CON) compared to control mice (CON) on day 20 (Table 2A). Thedifference in body weight change already being significant at day 15after tumor inoculation (Table 2C). Again, a significant lower fat mass(epididymal fat) and muscle mass was observed in the TB-CON mice (Table2B). Food intake was not different between groups (Table 2D). Thetumor-bearing mice receiving the Specific Nutritional Combination(TB-SNC group) had a higher body weight, and delta body weight comparedto TB-CON mice. The attenuation of body weight loss in the TB-SNC micecoincided with a reduction of fat loss and a reduction in muscle wasting(mTA, mG, and mS) (Table 2B). Organ (wet) mass of kidney, liver, thymusand heart either decreased with increased cachexia or showed no change.Nutritional supplementation resulting in increased carcass weight partlycompensated the weight loss. For experiment B, the data for organ masses(in percentage of control (CON)±SEM) were: kidney: TB-CON: 81%±2;TB-SNC: 91%±2, liver TB-CON: 88%±2; TB-SNC: 92%±3, thymus TB-CON: 46%±4;TB-SNC: 55%±4, heart TB-CON: 86%±2; TB-SNC: 88%±2, and lung: TB-CON:98%±2; TB-SNC: 103%±3. Tumor mass was not increased by any of thenutritional supplementations (Tables 1 and 2).

TABLE 2 Cachexia parameters as a result of an intervention with thespecific nutritional combination. A Body, tumor and carcass weight atsection (g at day 20) treatment N BW delta BW TW CW delta CW CON 10 28.0± 0.7 *  5.3 ± 0.5 * 0.0 ± 0.0 * 28.0 ± 0.7 *  5.3 ± 0.5 * TB-CON 1720.8 ± 0.5 −0.7 ± 0.4 2.1 ± 0.1 18.7 ± 0.4 −2.8 ± 0.4 TB-SNC 18 23.1 ±0.6 *  0.9 ± 0.6 * 1.7 ± 0.1 * 21.4 ± 0.6 * −0.7 ± 0.7 * B Organ weightsat section (mg at day 20) treatment N Epididymal fat mTA mG mEDL mS CON10 443 ± 37 * 44.5 ± 1.3 * 141 ± 4 * 9.2 ± 1.1 6.7 ± 0.5 * TB-CON 17  87± 18 33.4 ± 0.9 108 ± 2 7.8 ± 0.2 5.3 ± 0.2 TB-SNC 18 189 ± 20 * 38.1 ±0.9 * 118 ± 3 * 8.3 ± 0.5 5.7 ± 0.2 * C Change in BW in time change inBW (change in g: day 0-20 when compared to day −1) treatment N 0 6 10 1520 CON 10 1.0 ± 0.3 3.6 ± 0.4 4.3 ± 0.7 5.5 ± 1.0 *  6.3 ± 1.4 # TB-CON17 0.6 ± 0.3 2.7 ± 0.3 3.3 ± 0.3 3.7 ± 0.4 −0.1 ± 0.5 TB-SNC 18 0.9 ±0.2 3.4 ± 0.3 4.4 ± 0.4 4.6 ± 0.4  1.8 ± 0.6 # D Foodintake in timeFoodintake per day (g) treatment N 8 13 17 19 CON 10 4.4 ± 0.2 4.3 ± 0.24.4 ± 0.3 3.5 ± 0.6 TB-CON 17 4.0 ± 0.2 4.1 ± 0.1 3.7 ± 0.3 3.5 ± 0.5TB-SNC 18 4.2 ± 0.1 4.5 ± 0.2 4.8 ± 0.3 4.4 ± 0.4 CON = mice receivingcontrol diet B, TB-CON = tumor-bearing mice receiving control diet B,TB-SNC = tumor-bearing mice receiving the specific nutritionalcombination. BW = body weight; delta BW = BW day 20 minus BW day 0,delta CW = CW day 20 minus CW day 0, TW = tumor weight, mTA = muscleTibialis Anterior, mG = muscle Gastrocnemius, mEDL = muscle ExtensorDigitorum Longus, mS = muscle Soleus. Data as means ± SEM: * =significantly different from TB-CON (p < 0.05, k = 2, α = 10%); # =significantly different from TB-CON for the whole curve (p < 0.05, k =2, α = 10%). (For more details about statistics, see the Materials andMethods).

Ex-Vivo Muscle Function (Category B Experiment).

Force-frequency characteristics (10 to 167 Hz, 250 ms) were determinedex vivo in mEDL. Maximal force, maximal contraction velocity and maximalrelaxation velocity were significantly different in TB-CON when comparedto CON and TB-SNC (FIGS. 2 A, B, and C). When these parameters werecorrected for muscle mass, overall curve positions maintained.Significant differences, however, only remained between CON and TB-CON.To further investigate muscle mass-independent changes in musclefunction the time needed for a contraction (CT90) was determined. CT90was defined as the time needed to go from 10 to 90% of maximalcontraction force, at frequencies at which tetanus was obtained. CT90was significantly different between TB-SNC and TB-CON at lowerfrequencies at which total tetanus could be obtained (83 and 100 Hz).These data suggest that at frequencies (83-100 Hz) relevant forefficient physical performance (tetanus present), besides musclemass-dependent changes, also muscle mass-independent changes hadoccurred that were corrected by specific nutritional intervention.Therefore, an exercise protocol of 100 repeated pulses was applied at 83Hz. Again, CON and TB-SNC were significantly different from TB-CONduring the whole exercise protocol for maximal contraction force (FIG.3A) and maximal contraction velocity (FIG. 3C). When maximal contractionforce was corrected for muscle mass (FIG. 3B) curve positions remained,with only significant differences between CON and TB-CON. Maximalcontraction velocity of the TB-SNC group, however, was stillsignificantly different from TB-CON when corrected for muscle mass inthe first repeats of the exercise (<10 repeats) (FIG. 3D).

Physical Activity (Category B Experiment).

Total daily activity showed a significant interaction between time andgroup (P<0.01; RM-ANOVA) over the total period (2-19 days). Activitylevels in TB-CON mice were significantly lower than in control mice ondays 10-11 (P<0.05), and from day 16 onwards (P<0.01). The TB-SNCanimals did not differ significantly from the control animals in theirtotal activity throughout the experiment, while their activity wassignificantly higher at days 18-19 compared with TB-CON mice (P<0.05)(FIG. 4A). These differences in total activity resulted from significantchanges during their active period (i.e. dark period) (FIG. 4B).Throughout the dark period, TB-CON mice were significantly less activethan controls on days 16-17 and 18-19 (P<0.01), resulting in a drasticdecrease in overall activity in the TB-CON mice. The TB-SNC mice wereless active than control mice during the dark on day 18-19 (P<0.05), butmore active than TB-CON mice on those days (P<0.05).

Besides a reduction in daily activity level of TB-CON mice, a clearshift in daily activity pattern was observed, i.e. from dark to light,both in tumor-bearing controls and in TB-SNC animals on days 18-19 (FIG.4C). To focus on possible shifts in daily activity pattern, hourlyactivity pattern during the day was expressed as a percentage of thetotal (100%) daily activity on that specific day (FIG. 4D) (i.e. notreferring to day 2 and not corrected for the graduate decline inactivity for the tumor-bearing groups). At baseline (days 2-3) allgroups showed comparable day/night rhythms. Animals were active duringthe dark and had an inactive period during the light. A relative shifttowards increased activity during the light period is observed in theTB-CON group from day 16, which occurs less or delayed in the TB-SNCgroup.

Discussion

The present study comparing nutritional intervention with single andmultiple components clearly supports the added value of a multi targetapproach with specific nutrients on body composition in the murine C26carcinoma model. In addition, the specific nutritional combination alsoimproved muscle function. Moreover, activity patterns as well as overalldaily activity improved, probably as a consequence of improved bodycomposition and muscle function. These findings are highly relevant tothe clinical situation, because muscle function and daily activity areimportant contributors to the quality of life of the cancer patient.Therefore, these data strongly endorse the use of specific nutritionalsupport for cancer patients with a combination of multiple ingredients.

The data focus on the specific nutritional needs of the cancer patientto improve or prevent cachexia characteristics, and show the effects ofdifferent isocaloric nutritional interventions with single ingredientsor combined nutritional components in the C26 murine model ofcancer-induced cachexia. There were no significant differences on foodintake between groups on complete curves nor on analysis per day up today 19. These data confirm earlier findings that the C26 adenocarcinomamouse model is a cachectic non-anorectic model. The observation,however, that in experiment A, food intake of Con is significantlyhigher than that of TB mice on day 20 specifically indicates that iftumor growth would continue for a few days more, the tumor bearinganimals would likely become anorectic. The differences in cachecticparameters between control and tumor-bearing mice 20 days after tumorinoculation, were comparable in magnitude to those described in otherstudies also using the C26 adenocarcinoma mouse model.

From the single components only fish oil increased fat mass. Fat masshas been suggested to be important in survival of the patient whilemuscle mass has been implicated to contribute specifically to thequality of life of the patient. The data on mTA muscle mass show that atthe tested concentration the combination of all components, i.e. fishoil, high protein and leucine were needed for a significant effect onmuscle mass (FIG. 1). These results are in line with the hypothesis thatnext to an increase in anabolic responses, protein catabolism has to bedecreased via reduction of inflammation to reach a positive effect onmuscle protein mass in a cancer cachectic state. There is growingsupport that the inflammatory response to a tumor attributesconsiderably to the progression towards cachexia. It has also beensuggested that the increase in catabolic vs. anabolic processescontributes to the failure to accumulate lean body mass even whennutritional intake is normal. Clinical data from different cachecticpatient groups in the literature suggest that fish oil might reducecatabolism and weight loss. Fish oil probably not only attenuates thetumor-induced inflammatory response, but also normalizes the insulinresistance present in the cachectic state. Prolongation of survival hasbeen reported in a mixed group of advanced cancer patients supplementedwith ω-3 fatty acids and vitamin E (Gogos, C. A., Ginopoulos, P., Salsa,B., Apostolidou, E., Zoumbos, N. C., and Kalfarentzos, F. Cancer, 82:395-402., 1998) which might also result from immune-modulation. Thesuggestion that in cachectic patients fish oil might contribute to themaintenance of body composition via a reduction of inflammatoryresponses, is supported by our data. High protein with leucine(hpr+leu), did not result in significant changes in mTA mass. However,when fish oil was added, the combination of high protein and leucine(hpr+leu+fo) contributed to a significant weight gain of mTA. Therefore,it is hypothesized that reduction of the inflammatory state by fish oilimproved the sensitivity of the animals to anabolic stimuli like leucineand high protein, resulting in improved maintenance of muscle proteinmass.

Results from in vivo studies suggest that BCAAs and especially leucineregulate skeletal muscle protein metabolism (Rooyackers, O. E. and Nair,K. S. Annu Rev Nutr., 17: 457-485., 1997). This signal is related toactivation of the mTOR pathway. In healthy volunteers, leucine has beenreported to provide a signal for stimulation of muscle protein synthesisand to possibly decrease muscle protein breakdown (Rennie, M. J., Bohe,J., Smith, K., Wackerhage, H., and Greenhaff, P. J Nutr., 136:264S-268S., 2006). In healthy individuals this signal is likely to beshort-lived due to the ‘muscle-full phenomenon’ induced by normalnutritional intake and homeostatic control mechanisms. In contrast, longlasting effects of BCAA supplementation were reported in patients with ametabolic or nutritional deficiency like in septic or cancer patients.In these patient groups BCAA supplementation was reported to result inpositive effects on albumin status, quality of life and overallsurvival. Moreover, it has been reported that protein synthesis can onlybe stimulated in the presence of a high supply of balanced amounts ofessential amino acids (Rooyackers, O. E. and Nair, K. S. Annu Rev Nutr.,17: 457-485., 1997). Altogether, these studies suggest that acombination of high protein and BCAA supplementation might result inimproved protein metabolism, resulting in muscle mass gain, which couldcontribute to a lower morbidity and a higher quality of life. Our dataindeed suggest that both leucine and high protein supplementationcontribute to the cumulative effect on muscle mass maintenance, reachedby the total nutritional combination (FIG. 1 and Table 1).

To our suprise, a combination of supplementation of high protein,leucine and fish oil resulted in a surplus value with respect to a broadspectrum of parameters characterizing cachexia. The group in which allnutritional components were combined (TB+hpr+leu+fo mice) was the onlygroup that showed significant differences versus TB on all read-outparameters of cachexia (e.g. weights of body, carcass, muscles and fat(see Table 1). The suggested additive effects of single nutritionalcomponents to the total combination are best illustrated by the data ontibialis muscle mass (mTA FIG. 1). These data clearly indicate a surplusvalue of a multi-nutritionalcomponent approach. We suggest that theobserved additive effects originate from presumed differences inmechanistic targets of these components, i.e. 1) stimulation of anabolicsignals by supplementation of building blocks (essential amino acids)and by stimulation of mTOR (leucine), 2) reduction of protein catabolismby the reduction of inflammatory and hormonal responses (fish oil) anddown regulation of the signaling pathway leading to protein breakdown(leucine), and the possible interaction(s) between these mechanisms. Thesecond experiment confirmed the efficacy of the nutritional combinationon body composition maintenance. Moreover, in this experiment thecombination of ingredients also improved parameters reflecting physicalperformance like muscle function and daily activity patterns.

Organ (wet) mass of kidney, liver, intestine, thymus and heart wereunaffected or decreased with increased cachexia. Nutritionalsupplementation resulting in increased carcass weight had no effect orpartly normalized the loss in organ mass. Moreover, none of the selectedingredients increased tumor size. The complete nutritional combinationshowed a reduction in tumor size in experiment B.

C26-tumor inoculation induced a loss of muscle function. A large part ofthe reduction of muscle function was explained by a reduction in musclemass. These findings are in accordance with clinical data. Gogos et alreported a significantly higher Karnofsky performance status inmalnourished patients supplemented for 40 days with 18 g of ω-3 PUFAcompared with placebo (Gogos, C. A., Ginopoulos, P., Salsa, B.,Apostolidou, E., Zoumbos, N. C., and Kal-farentzos, F. Cancer, 82:395-402., 1998.). These data suggest that improvement of physicalactivity may occur even before a significant weight gain is achieved.This may indicate that for maintaining normal life activities,preventive treatment to reduce muscle wasting is recommended. In ourexperimental setup, all tumor-induced muscle mass-dependent decreases inmuscle function could be significantly restored by supplementation withthe specific nutritional combination. These data are supported byclinical trial data of Barber et al. reporting an improved functionalperformance after 3 and 7 weeks of supplementation with 2.2 g EPA+0.96 gDHA in unrespectable pancreatic cancer patients. The improved physicalperformance coincided with increased BW and appetite (Barber, M. D.,Fearon, K. C., Tisdale, M. J., McMillan, D. C., and Ross, J. A. NutrCancer, 40: 118-124, 2001.). Next to muscle mass-dependent changes inmuscle function, also muscle mass-independent loss of function issuggested by presented data (FIGS. 2D and 3D). A tumor-related, musclemass independent decrease in muscle function has not been describedbefore. This compromised muscle function became especially manifest inthe maximal contraction velocity after exercise of moderate strength(FIG. 3C). The muscle mass-independent decrease in muscle function couldalso be partly restored by supplementation with the specific nutritionalcombination (TB-SNC). These results indicate that the nutritionalcombination restores both muscle mass dependent and muscle massindependent decreases in muscle function.

Asthenia, resulting from cancer cachexia, leads to a reduced dailyactivity. Indeed, in the presence of a tumor, daily activity levels ofmice decreased over time which is in line with clinical reports ofcancer patients. It is not clear what mechanism induces the reduction inactivity in cachectic cancer patients. Reduced muscle mass and decreasedmuscle force may contribute to the deterioration in activity. Inaddition, the tumor-induced inflammatory response might further reducethe daily activity. Physical activity is a major determinant of qualityof life (Moses, A. W., Slater, C., Preston, T., Barber, M. D., andFearon, K. C. Br J Cancer, 90: 996-1002, 2004). The complete nutritionalcombination tested maintained activity compared to TB-CON mice. Thiseffect may be directly related to the better maintained physicalperformance (improved muscle mass and function). The influence of thenutritional combination on other factors involved in physicalperformance, however, would need further examination.

Chevalier et al. reported that patients with advanced colorectal cancershowed less contrast between day time and night time activity (nocturnalsleep) (Chevalier, V., Mormont, M. C., Cure, H., and Chollet, P. OncolRep, 10: 733-737, 2003.). Individual activity patterns have even beensuggested to be predictive of the patients' survival, tumor response andquality of life. The possibility of a tumor-induced disturbance indiurnal activity patterns is supported by our data, indicating atumor-related shift in activity from the dark to the light period. Thespecific nutritional combination tested shows a clear trend to reducethis effect. Normal sleep patterns are critically dependent on thecircadian release of melatonin from the pineal gland. DHA-enrichedformulas have been reported to normalize melatonin secretion in(ω-3)-deficient rats (Zaouali-Ajina, M., Gharib, A., Durand, G., Gazzah,N., Claustrat, B., Gharib, C., and Sarda, N. J Nutr, 129: 2074-2080,1999.); this might also be an explanation for the results obtained inour experiments.

Based on the results from this study, it is clear that more attentionshould be paid to prevention of cachexia in order to maintain quality oflife for the patient.

In conclusion, a nutritional combination of high protein, leucine andfish oil improved the cachectic outcome of mice inoculated with the C26adenocarcinoma cell line. Not only did the carcass, fat and muscle massincrease, also muscle function and daily activity improved when comparedto tumor-bearing mice on the control diet. These data show that singleingredient interventions have limited value, and support the need for abalanced combination of different ingredients to enable a multi-targetedintervention to achieve effects in the complex conditions of cancercachexia.

Example 2: Formulation Examples

A sip feed may in particular comprise macronutrients in ranges specifiedin Table 3. A specific example is given in Table 4. In addition one ormore micronutrients (such as minerals, vitamins, etc.) and/or one ormore other food-grade additives (e.g. flavourings; preservatives;nonproteinogenic amino acids, such as carnitine) may be present.

TABLE 3 Nutritional composition of a Sip Feed (per 100 ml) Proteinaceousmatter (equivalent) (g)  9-12 containing total whey protein 1-9containing total leucine (g) 1.5-2.5 of which Leucine as free amino acid(g) 0.9-1.5 Carbohydrate (g) 10-25 Fat (g) 2-6 of which unsaturated 2-6of which omega 3 poly unsaturated 0.8-2  of which EPA, DHA, ETA, DPA0.2-2  Soluble fibre, dietary g 1-4

TABLE 4 Nutritional composition of a sip feed (per 100 ml) Proteinaceousmatter (equivalent) (g) 10.1 containing total whey protein 2.9containing total leucine (g) 2.0 of which Leucine as free amino acid (g)1.1 Carbohydrate (g) 17.4 Fat (g) 5.3 of which unsaturated 4.2 of whichomega 3 poly unsaturated 1.1 of which EPA, DHA, ETA, DPA 1.1 Solublefibre, dietary g 2.0

A tube feed may in particular comprise macronutrients in rangesspecified in Table 5. A specific example is given in Table 6. Inaddition one or more micronutrients (such as minerals, vitamins, etc.)and/or one or more other food-grade additives (e.g. flavourings,preservatives) may be present.

TABLE 5 Nutritional composition of a tube feed Proteinaceous matter(equivalent) (g)  6-10 containing total whey protein 1-8 containingtotal leucine (g) 1-2 of which Leucine as free amino acid (g) 0.4-0.9Carbohydrate (g) 10-25 Fat (g) 2-6 of which unsaturated 2-6 of whichomega 3 poly unsaturated 0.4-1  of which EPA, DHA, ETA, DPA 0.1-1 Soluble fibre, dietary g 1-4

TABLE 6 Nutritional composition of a tube feed Proteinaceous matter(equivalent) (g) 7.8 containing total whey protein 2.9 containing totalleucine (g) 1.5 of which Leucine as free amino acid (g) 0.8 Carbohydrate(g) 17.2 Fat (g) 5.4 of which unsaturated 4.5 of which omega 3 polyunsaturated 0.8 of which EPA, DHA, ETA, DPA 0.7 Soluble fibre, dietary g1.5

The following composition (Table 7) was made according to standardprocedures and is suitable for use according to the invention,preferably as a sip feed.

TABLE 7 Main ingredients of a specific composition according to theinvention INGREDIENTS AMOUNT Energy content 160 kcal/100 ml Protein (27en %) 10.1 g/100 ml of which: whey: 2.9 g/100 ml casein: 6.1 g/100 mladded leucine: 1.1 g/100 ml wherein the following amino acids arepresent (based on total protein weight): L-Leucine: 19.4 wt %L-Glutamine/Glutamic acid: 17.8 wt % L- Cysteine: 0.9 wt % Lysine: 7.5wt % leu/(val + ile) − ratio = 1.83 Carbohydrates (43 en %) 17.4 g/100ml of which: sugar blend comprising glucose, galactose, lactose,maltose, sucrose and trehalose (12.7 g/100 ml) starch (4.3 g/100 ml)Lipids (30 en %) 5.3 g/100 ml of which: ω-3 ALA (1.8 g/100 g of totallipid) EPA (11.9 g/100 g of total lipid) DHA (5.8 g/100 g of totallipid) DPA (1.4 g/100 g of total lipid) SDA (1.8 g/100 g of total lipid)ω-6 LA (26.0 g/100 g of total lipid) AA (0.7 g/100 g of total lipid)ω-3/ω-6 = 0.87 Others: Dietary fiber 2 g/100 ml ofgalactooligosaccharides L-carnitine 10.9 mg/100 ml Taurine 13.2 mg/100ml Viscosity 41 mPa · s

Example 3: Muscle Protein Synthesis after Nutritional Supplementation inColorectal Cancer Patients

A sip feed containing a specific nutritional combination as described inTable 4 from the possible compositions was tested on its capacity toinfluence muscle protein synthesis rate and compared to a controlproduct as described in Table 8.

TABLE 8 Supplements Composition Specific Nutritional Combination (SNC)(100 ML) CONTROL (100 ML) Energy Kcal 160 Energy Kcal 160 Protein* g10.1 g Protein* g 6.0 Fat g 5.3 Fat g 5.8 Carbohydrates g 17.4Carbohydrates g 21.0 *SNC: 2.9 g whey protein, 1.1 g free leucine,balance casein; control only casein

Research Design and Methods

Study Subjects.

Subjects were enrolled based on the inclusion/exclusion criteriadescribed below. All subjects were able to walk, sit down and stand upon their own. Screening procedures not already performed in the contextof their care for cancer was done prior to the study. A total of 24subjects (12 each group) completed the protocol. The inclusion criteriawere as follows: (1) Radiographic evidence of cancer, (2) Age >40 years(both male and female), (3) Ability to sign informed consent.

Overview of the Study Design.

A randomized, controlled, double-blind, parallel-group design in 24patients with recently diagnosed metastatic colorectal cancer wasutilized. Subjects were initially interviewed, and the experimentalprocedures were explained in detail and signed, informed consentsobtained. After acceptance into the study, subjects received all oftheir meals for 3 days prior to the experimental phase of the study tostandardize food intake. The meals were prepared to be taken home to beeaten. The evening before the study the subjects refrained from any foodor drink (except water) intake from 22:00 hr onwards. The experimentalphase of the study started the following morning and lasted forapproximately 10 hours. Twelve patients ingested the sip feed containingthe specific nutritional combination (SNC) and the other 12 patientsingested a control supplement (CS). Each subject ingested 400 ml of thesupplements in two doses. The intake of the second dose started twentyminutes after the first sip of the first dose. Each dose was consumedwithin 10 minutes. Subjects were randomly assigned with respect to thesupplement to be ingested, stratified for gender.

Experimental Procedures.

On the morning of the study two 18-22 gauge catheters were placed by thestudy nurse into veins of the right and left forearms, and one used forblood sampling and the other for tracer infusion. After obtaining ablood sample for background amino acid enrichment and fasting bloodglucose, a priming or “loading” (2 μmol/kg) infusion ofU-13C6-phenylalanine was given. This was immediately followed by acontinuous (0.07 μmol/kg/min) infusion of U-13C6-phenylalanine andmaintained throughout the experiment. A muscle biopsy was performed at 2hours after the start of isotope infusion and again at 5 hours. Bloodwas also taken from the sampling forearm catheter periodically for thedetermination of amino acid enrichments (plasma tracer/tracee ratio).Immediately following the second muscle biopsy, one dose of thesupplement (200 ml) was given, followed by a second dose (200 ml) 20minutes after the first sip of the first dose. Each dose was consumedwithin 10 minutes. The third muscle biopsy was taken 300 minutes afterthe first sip of the first dose of supplement. The subjects were lyingin bed throughout the study unless they had to use the bathroom. Musclebiopsies were used to calculate muscle protein fractional synthetic rate(FSR). Plasma samples were analyzed for amino acid enrichments (plasmatracer/tracee ratio) glucose and amino acid concentrations.

Results.

Muscle protein synthesis was similar at base line for SNC and controlgroup (each n=12 patients) (see FIG. 5). Ingestion of 400 ml of controlsip resulted in a fractional synthesis rate similar to baselinesynthesis rate, while supplementation with the specific nutritionalcombination according to the invention (SNC), resulted in an increase infractional synthesis rate of 1.4 times the fractional synthesis rate atbaseline.

Glucose concentrations were reduced in patients receiving thecomposition according to the invention (SNC) when compared to patientsreceiving the control supplement (FIG. 6). This is advantageous becauselow post prandial glucose levels can (on term) result in improvedsensitivity of the muscle for an anabolic triggers (insulin).

Example 4: Glycemic Index Determination

Definition. The glycemic index (GI) of a carbohydrate provides a measureof its ability to raise postprandial glucose concentrations. High GIfoods give higher postprandial blood glucose levels than those with alow GI. The GI of a carbohydrate also predicts the insulin response tothat food.

The GI of a carbohydrate is calculated by assessing a 25 gtwo-hourglycaemic response with that of a subsequent 25 g carbohydratestandard glucose:

GI equals ‘Incremental area under blood glucose response curve for atest food containing 25 g of carbohydrate’ divided by ‘Correspondingarea after equivalent carbohydrate portion of glucose’

Glycaemic Index Methodology.

Available carbohydrate is defined for GI testing purposes as: Totalcarbohydrate minus the indigestible carbohydrates (soluble andinsoluble) that are from a physiological point dietary fibres (e.g.inulin, FOS, type 3 resistant starch).

The samples provided should be representative of the product asavailable to the consumer in the market place.

All foods submitted for testing are tested in vivo, that is, in 10 humansubjects consuming amounts containing the equivalent of 25 g availablecarbohydrate. They are healthy subjects with no chronic diseases,diabetes or glucose impairment. Subjects have a BMI between 18.5-27kg/m².

Reference food: The reference food is 25 g glucose powder dissolved in250 mls water. Each person tests the reference food at least twice.

Test foods: The test foods are prepared according to manufacturer'sinstructions, representing the food as normally consumed. The test foodsare consumed once only on separate occasions as a portion providing 25 gof available carbohydrate, defined as above.

Protocol Subjects: Subjects are tested in the morning after a 10-12 hovernight fast. Two fasting blood samples are taken (−5 & 0) 5 minutesapart after which subjects consume the test meal or reference food at aneven rate over 15 minutes. Further blood samples are taken at 15, 30,45, 60, 90 and 120 minutes after the beginning of the meal. The testmeal and reference food should be consumed with a 250 mls drink ofwater. This remains constant for each of the tests in the series.

24 hrs prior to GI test: The day before each session, subjects refrainfrom drinking alcohol and avoid unusual levels of exercise and foodintake. Subjects must have an evening meal based on a carbohydrate-richfood, such as rice, pasta, bread, potatoes and not too much fat. Thismeal should not include beans, pulses or legumes (to avoid a second mealeffect the next morning). It is important that they eat dinner and notfast for more than 18 hours. Subjects are asked to be in a similar stateeach time they come in for a session. After they have eaten theirevening meal, subjects fast for at least 10 hours overnight before thestart of their test session the next morning. They can drink only waterduring the fasting period.

Blood sampling: Blood will be obtained by finger pricking.

Blood is collected without clotting inhibitors (heparin, EDTA).

Glucose assay: Whole capillary blood or is measured by an automaticglucose analyzer. In this case, Hemocue glucose analysers are used.

Data analysis: The incremental area under the blood glucose responsecurve (iAUC), ignoring area beneath the baseline, is calculatedgeometrically as follows:

For times t0, t1, . . . tn the blood glucose concentrations are G0, G1,. . . Gn, respectively:

${iAUC} = {\sum\limits_{n}^{x = 1}{Ax}}$

-   -   wherein Ax=the AUC for the xth time interval (ie. between tx−1        and tx).    -   For the first time interval (ie. x=1): if G1>G0,        A1=(G1−G0)×(t1−t0)/2 otherwise, A1=0

For the other time intervals (ie. x>1)

if Gx≧G0 and Gx−1≧G0, Ax={[(Gx−G0)/2]+(Gx−1−G0)/2}×(tx−tx−1)

if Gx>G0 and Gx−1<G0, Ax=[(Gx−G0)²/(Gx−Gx−1)]×(tx−tx−1)/2

if Gx<G0 and Gx−1>G0, Ax=[(Gx−1−G0)²/(Gx−1−Gx)]×(tx−tx−1)/2

if Gx<G0 and Gx−1<G0, Ax=0

GI calculation: In individual subjects, the GI value is the iAUC foreach food expressed as a percentage of the mean iAUC of the tworeference foods (glucose). The GI of the test food is the mean GI±SEM ofthe 10 subjects.

Up to two outliers (an outlier is an individual whose GI differs fromthe mean by more than two SD) may be excluded from the data set. SEMshould be within 20% of the mean.

Clinical Trial Setting 1: Healthy Volunteers.

Ten healthy subjects having a BMI between 18.5-27 kg/m2, were testedafter a 10-12 h fast. Each subject was tested in a cross over design,receiving 25 g of carbohydrates from a standard reference glucose drink(2 times), a standard sip (16 EN % protein, 50 EN % carbohydrates and 34EN % fat) or the test drink according to the invention (“SNCGI value wascalculated as the incremental area under the blood glucose responsecurve for each food (iAUC) as a percentage of the mean iAUC of thereference glucose drink.

Results.

The GI for the standard sip was 67±10 whereas the GI for the test drinkwas 40±4. Therefore the GI of Forticare was classified as low (<55) andthe standard sip as medium (55-70).

Clinical Trial Setting 2: Cancer Patients:

As described in Example 3 Glucose concentrations were reduced inpatients receiving the specific nutritional combination SNC whencompared to patients receiving the control supplement (Example 3, FIG.5).

1.-32. (canceled)
 33. A method for improving the muscle function of amammal, comprising administering to said mammal a nutritionalcomposition comprising at least 18 en % of proteinaceous matter having aleucine content of at least 9.5 wt % based on total proteinaceousmatter, a lipid fraction comprising at least one ω-3-polyunsaturatedfatty acid selected from the group consisting of eicosapentaenoic acid(EPA), docosahexaenoic acid (DHA), eicosatetraenoic acid (ETA) anddocosapentaenoic acid (DPA).
 34. The method according to claim 33,wherein said proteinaceous matter comprises whey and at least 12 wt % ofleucine, based on total proteinaceous matter.
 35. The method accordingto claim 33 wherein the muscle function is skeletal muscle function. 36.The method according to claim 35, wherein improving skeletal musclefunction comprises improving muscle mass dependent loss of musclefunction comprising a correction in maximal force, maximal contractionvelocity or maximal relaxation velocity of skeletal muscle.
 37. Themethod according to claim 35, wherein improving skeletal muscle functioncomprises improving muscle mass independent loss of muscle functioncomprising a correction in maximal force corrected for muscle mass,maximal contraction velocity corrected for muscle mass or maximalrelaxation velocity corrected for muscle mass.
 38. The method accordingto claim 33, wherein improving the muscle function is directed atpreventing or treating a reduction of muscle function due to, orresulting from aging, disease, disorder, drugs or trauma, preferablydrug, disease or disorder.
 39. The method according to claim 38, whereinthe disease or disorder is a cancer.
 40. The method according to claim38, wherein the drug is administered in the framework of a chemotherapy.41. The method according to claim 33, wherein the nutritionalcomposition is a liquid, comprising at least 7 g/100 ml of proteinaceousmatter.
 42. The method according to claim 33, wherein the proteinaceousmatter further comprises at least one protein from a protein sourceselected from the group consisting of casein, caseinate, soy and wheat.43. The method according to claim 33, wherein the proteinaceous mattercomprises at least 25 wt % of whey, based on the total proteinaceousmatter.
 44. The method according to claim 33, wherein the compositioncomprises leucine in the form of a free acid, a salt, a dipeptide or aconjugate with a conjugating compound other than an amino acid, aprotein, or a peptide, which conjugate is capable of being split intothe free amino acid or salt thereof in the gut or stomach or afterabsorption in the enterocytes or liver.
 45. The method according toclaim 33, wherein the composition comprises 12 to 23 wt % of leucine,based on total proteinaceous matter.
 46. The method according to claim33, wherein the composition comprises carnitine.
 47. The methodaccording to claim 33, wherein the composition comprises taurine. 48.The method according to any claim 33, wherein the composition comprisesat least 15 wt % of an ω-3 polyunsaturated fatty acid, based on totallipid content.
 49. The method according to claim 33, wherein the lipidfraction comprises less than 30 wt % of a saturated fatty acid, based ontotal lipid content.
 50. The method according to claim 33, wherein thelipid fraction further comprises at least one ω-6 polyunsaturated fattyacid, and wherein the molar ratio of ω-3 polyunsaturated fatty acids toω-6 polyunsaturated fatty acids is less than 1.0.
 51. The methodaccording to claim 33, wherein the composition further comprises anindigestible carbohydrate, selected from the group consisting ofgalactooligosaccharides and fructooligosaccharides.
 52. The methodaccording to claim 33, wherein: a) the proteinaceous matter contentprovides 18 to 60 en %, said proteinaceous matter comprising whey; b)the lipid content provides 10 to 50 en %, of the total composition; c)the digestible carbohydrate content provides 20 to 70 en % of the totalcomposition.
 53. The method according to claim 33, wherein thecomposition is a liquid nutritional composition having an energeticvalue in the range of 0.3 to 3.0 kcal/ml.
 54. The method according toclaim 33, wherein the nutritional composition is a liquid, having anenergetic value in the range of 0.2 to 1.0 kcal/ml.
 55. The methodaccording to claim 33, wherein the nutritional composition is asemi-liquid, gel or solid, having an energetic value in the range of 3.2to 8.0 kcal/g.
 56. The method according to claim 33, wherein thenutritional composition further comprises at least one componentselected from the group consisting of minerals, trace elements andvitamins, preferably selected from the group consisting of sodium,potassium, chloride, fluoride, iodide, calcium, phosphorous, magnesium,vitamin A, vitamin D3, vitamin E, vitamin K, vitamin B1, vitamin B2,vitamin B3, vitamin B5, vitamin B6, folic acid, vitamin B12, biotin,vitamin C, lipoic acid, zinc, iron, copper, manganese, molybdenum,selenium and chromium.
 57. The method according to claim 33, wherein themethod further comprises improving daily activity, improving physicalperformance, providing a better prognosis in terms of extendedlife-expectancy, improving compliance to an anti-cancer therapy orimproving a quality of life.
 58. The method according to claim 33,wherein the lipid fraction further comprises at least one ω-6polyunsaturated fatty acid, and wherein the molar ratio of ω-3polyunsaturated fatty acids to ω-6 polyunsaturated fatty acids is from0.5 to 0.97.
 59. The method according to claim 33, wherein the lipidfraction further comprises at least one ω-6 polyunsaturated fatty acid,and wherein the molar ratio of ω-3 polyunsaturated fatty acids to ω-6polyunsaturated fatty acids is from 0.6 to 0.95.